GPCRs are transmembrane receptor proteins that are responsible for the transduction of a diverse array of extracellular signals, including hormones, neurotransmitters, peptides, lipids, ions, light, odorants, nucleotides, fatty acid derivatives, and other chemical mediators. See, e.g., WO 2002/00719. GPCRs are of particular importance to drug discovery because they have been established as excellent drug targets: they are the targets of 50% of marketed drugs. An increasing number of diseases have been found to be associated with GPCRs. Drugs targeting GPCRs have been used to treat a wide range of disorders from cardiovascular to gastro-intestinal to CNS and others (Wilson et al., 1998, British J. of Pharmacology, 125:1387-1392).
The GPCR-mediated signal transduction event is often initiated upon binding of a specific ligand to the GPCR. Each GPCR is composed of an extracellular N-terminal domain, seven distinct transmembrane segments, and an intracellular C-terminal domain. Binding of the ligand to an extracellular N-terminal domain, transmembrane domain, or intracellular loop of a GPCR results in a conformational change that leads to activation of intracellular heterotrimeric GTP-binding proteins (G proteins) associated with the GPCR. These activated G proteins in turn mediate a variety of intracellular responses that regulate cell physiology. Therefore, the ligand provides means of elucidating the physiological function of the GPCR as well as methods of screening for compounds that regulate the signal transduction activity of the GPCR.
At present, only about two hundred GPCRs are classified as known GPCRs that are activated by around seventy known ligands. Through sequence analyses, it was discovered that GPCRs belong to one of the largest superfamilies of the human genome: evaluated at over one thousand genes encoding GPCRs (Civelli et al., 2001, Trends in Neurosciences, 24:230-237). A large number of putative GPCRs are described as orphan receptors because their natural ligands are unknown. Some of these uncharacterized orphan GPCRs may be useful as therapeutic targets. The identification of the specific ligand to a GPCR is the key to harnessing the potential therapeutic benefits of these orphan GPCRs (Howard et al., 2001, Trends in Pharmacological Sciences, 22:132-140).
One GPCR of interest is GPCR135, also known as SALPR (Matsumoto et al., 2000, Gene, 248:183-189). Relaxin-3 (also known as INSL7) has been found to be a ligand for GPCR135 as well as for GPCR142. See Liu et al., 2003a, Journal of Biological Chemistry, 278:50754-50764; Liu et al., 2003b, Journal of Biological Chemistry, 278:50765-50770; and International Publication Nos. WO 2004/082598 and WO 2005/014616. Relaxin-3 is a member of the insulin/relaxin superfamily. Members in this family are characterized by two peptide subunits (A-chain and B-chain) linked by three disulfide bonds. Two of the three disulfide bonds are inter-subunit bonds and another one is an intra-chain bond in the B-chain. In the family, insulin, IGF1, and IGF2 have been reported to be involved in the regulation of glucose metabolism and signal through tyrosine kinase/growth factor receptors, which are single transmembrane receptors. Another member of the relaxin/insulin superfamily is Insulin-Like (INSL) 5 (Conklin et al. 1999, Genomics, 60(1):50-56), which is believed to be a selective ligand for GPCR142 (see, e.g., U.S. Provisional Application No. 60/580,083, the disclosure of which is incorporated by reference herein). Two other members in the family are relaxin and INSL3, ligands for LGR7 and/or LGR8, which are GPCRs with leucine-rich repeats at the N-terminal extra-cellular domain. See also Hudson et al., 1983, Nature, 301:628-631; Hudson et al., 1984, EMBO J., 3:2333-2339.
Relaxin-3, a member of the insulin-relaxin peptide family (Bathgate et al., 2002, J. Biol. Chem. 277:1148-1157), and its receptor, GPCR135, are predominantly expressed in the brain (Burazin et al., 2002, J. Neurochem. 82:1553-1557). GPCR135 is expressed in many regions of the rodent brain, such as the superior colliculus, sensory cortex, olfactory bulb, amygdala and PVN (see, e.g., Sutton et al., 2004, Neuroendocrinology 80:298-307), suggesting potential physiological involvement in neuroendocrine and sensory processing. In vivo studies have further shown that relaxin-3 and GPCR135 are involved in stress response and in the regulation of feeding. Water restraint stress or intracerebroventricular (i.c.v.) CRF infusion induces relaxin-3 expression in cells of the nucleus incertus, where corticotrophin releasing factor receptor 1 is also expressed (Tanaka et al., 2005, Eur. J. Neurosci. 21:1659-1670). Central administration of relaxin-3 also induces feeding in rat (McGowan et. al., 2005, Endocrinology 146:3295-3300; Hida et al., 2006, J. Receptor and Signal Transduction 26:147-158).
In vitro, relaxin-3 activates receptors GPCR135, GPCR142, and LGR7 (Sudo et al., 2003, J. Biol. Chem. 278:7855-7862). The predominant brain expression of both relaxin-3 and GPCR135, coupled with their high affinity interaction, reflects that relaxin-3 is the endogenous ligand for GPCR135 (Liu et al., 2003a). In vitro pharmacological characterization, tissue expression profiling, and evolutionary study of GPCR142 and INSL5 indicate that GPCR142 is the endogenous INSL5 receptor (Conklin et al., 1999; Liu et al., 2003b; Chen et al., 2005, J. Pharmacol. Exp. Ther. 312:83-95). The high affinity interaction between relaxin and LGR7 as well as knockout studies demonstrate that relaxin is the endogenous ligand for LGR7 (Zhao et al., 1999, Endocrinology 140:445-453; Krajin-Franken et al., 2004, Mol. Cell Biol. 24:687-696).
Relaxin-3 activates not only GPCR135 and GPCR142, but also LGR7, which is expressed in both the brain and periphery (Hsu et al., 2000, Mol. Endocrinol., 14:1257-1271; Hsu et al., 2002, Science, 295:671-674; Tan et al., 1999, Br. J. Pharmacol., 127:91-98). The chimeric peptide R3/I5, composed of the relaxin-3 B-chain and the INSL5 A-chain, selectively activates GPCR135 over LGR7 (Liu et al., 2005b, Mol. Pharmacol., 67:231-240). See also WO 2006/026344.
Selective agonists having been discovered, there remains a desire to discover selective antagonists of GPCR135 and/or GPCR142 over LGR7. Since GPCR142 is a pseudogene in the rat (Chen et al., 2005) and is not detected in the mouse brain (Sutton et al., 2005, Neuroendocrinology, 82:139-150), activation of GPCR142 by central administration of relaxin-3 is not a great concern in murine species. However, potential activation of LGR7 by relaxin-3 remains problematic. LGR7 is expressed in the brain and is reported to play an important role in drinking (Thornton et al., 1995, J. Neuroendocrinol., 3:165-169; McGowan et. al., 2005) and potentially other physiological functions (Wilson et al., 2006, Neurosc.i Lett., 393:160-164; Nistri et al., 2005, Curr. Neurovasc. Res., 2:225-233; Sherwood, 2004, Endocr Rev., 25:205-234).